Gene structures of various living bodies as well as the functions of genes in genome scale have been widely studied. Accordingly, new technology for analyzing the gene functions has been rapidly developed.
A DNA micro-array (i.e., DNA chip) which is composed of a solid carrier (i.e., substrate) and a great number of spots of probe molecules such as DNA fragments or oligonucleotide molecules which are aligned and fixed on the solid carrier in separated areas is generally employed not only for base sequencing of a nucleic acid but also analyzing expression, mutation and polymorphism of gene. The analytical data of genetic information are favorably employable also for the study of pharmacologically active substances and further for diagnosis and prevention of diseases.
In the procedure for detection of a nucleic acid such as DNA fragment using the DNA micro-array, a nucleic acid sample (i.e., target nucleic acid) which is equipped with a radioisotope (RI) label or a fluorescent label is brought into contact with the probe molecules in the spots of the micro-array. If the target nucleic acid is complementary to the probe molecules in a certain base sequence, the target nucleic acid is combined with the probe molecules by hybridization. Thus hybridized target nucleic acid is detected by sensing its radioisotope label or fluorescent label. The results of the detection are then imagewise analyzed. The analytical procedure using the DNA micro-array can give a great number of data on the target nucleic acid simultaneously employing an extremely small amount of the target nucleic acid.
A DNA micro-array is prepared generally by synthesizing probe molecules (such as oligonucleotides) on a solid carrier (which is called “on-chip method”) or by fixing onto a solid carrier a number of previously prepared DNA fragments or oligonucleotides.
The former on-chip method is performed by synthesizing a number of oligonucleotides by combinatrial synthesis in each of extremely small areas predetermined on the solid carrier. In the synthetic procedure, photo-lithography and solid synthesis technology are utilized and a protective group is selectively removed by irradiation of light.
In the latter method, the previously prepared probe molecules such as DNA fragments or oligonucleotides are spotted on a solid carrier in each of the predetermined small areas and fixed onto the carrier by covalent bonding or ionic bonding (i.e., electrostatic bonding). The bonding is generally produced in the manner described below.
(1) In the case that the probe molecule is a DNA fragment such as cDNA fragment (i.e., complementary DNA fragment which is synthesized using mRNA as template) or a PCR product (i.e., DNA fragment produced from cDNA by multiplication procedure), an aqueous solution of the DNA fragments is spotted on a solid carrier having a coat of a polycation compound (such as poly-lysine or polyethyleneimine) by means of a spotting device of a micro-array preparing apparatus so that the DNA fragments can be electrostatically fixed onto the solid carrier utilizing electric charge of each DNA fragment.
(2) In the case that the probe molecule is a synthesized oligonucleotide, a reactive group is previously incorporated into the oligonucleotide. The oligonucleotide having the reactive group is then brought into contact with a solid carrier which has a reactive group on its surface in an aqueous medium using a spotting means so that the desired covalent bonding is produced between the reactive group of the oligonucleotide and the reactive group of the solid carrier. Examples of the reactive groups to be incorporated into the oligonucleotide include amino, aldehyde, mercapto (—SH), and biotin. On the surface of the solid carrier, a silane coupling agent having amino, aldehyde, epoxy, or the like is coated to incorporate the reactive group onto the surface. The fixation of oligonucleotide by covalent bonding is advantageous because it can produce bonding which is highly stable, as compared with the electrostatic bonding.
(3) In the case that the probe molecule is PNA (i.e., Peptide Nucleic Acid), a reactive group is previously incorporated into the probe PNA in the same manner as in the case (2) as above using the oligonucleotide.
It is preferred that the detection devices (e.g., DNA micro-array) prepared in the above-described preparing methods have probe molecules only on the solid carrier, because it electrostatic groups or reactive groups remain on the solid carrier with no cap groups, target molecules having a sensible label such as a fluorescent label may combine not only with the probe molecules but also with the electrostatic groups or reactive groups. The target molecules having been combined with the electrostatic groups and/or reactive groups produce noises in the analytical procedures.
It is already known to employ a blocking agent for obviating production of unfavorable combination between the target molecules and the electrostatic groups and/or reactive groups. The blocking agent can be employed by bringing the blocking agent into contact with a detection device having reactive groups and/or electrostatic groups on its surface. A representative blocking agent available on market is Denhaldt's solution. For blocking a detection device on which reactive groups remain on its surface, an aqueous monoethanol amine solution is known.
WO 97/14815 describes blocking a detection device using succinic anhydride or its analogue compound.